W Ian Deighan1, Valerie J Winton2, Rafael D Melani2, Lissa C Anderson3, John P McGee4, Luis F Schachner5, David Barnidge6, David Murray6, H Denis Alexander7, David S Gibson7, Michael J Deery8, Feargal P McNicholl9, Joseph McLaughlin7, Neil L Kelleher10, Paul M Thomas10. 1. Department of Clinical Chemistry, Altnagelvin Area Hospital, Londonderry, UK. 2. Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA. 3. Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, FL, USA. 4. Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA. 5. Department of Chemistry, Northwestern University, Evanston, IL, USA. 6. Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA. 7. Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute, Ulster University, Londonderry, UK. 8. Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK. 9. Department of Haematology, Altnagelvin Area Hospital, Londonderry, UK. 10. Proteomics Center of Excellence & Departments of Chemistry and Molecular Biology, Northwestern University, Evanston, IL, USA.
Abstract
OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.
OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.
Authors: Dirk Chelius; Kay Jing; Alexis Lueras; Douglas S Rehder; Thomas M Dillon; Alona Vizel; Rahul S Rajan; Tiansheng Li; Michael J Treuheit; Pavel V Bondarenko Journal: Anal Chem Date: 2006-04-01 Impact factor: 6.986
Authors: John P McGee; Rafael D Melani; Michael Goodwin; Graeme McAlister; Romain Huguet; Michael W Senko; Philip D Compton; Neil L Kelleher Journal: J Am Soc Mass Spectrom Date: 2020-02-24 Impact factor: 3.109
Authors: David R Barnidge; Surendra Dasari; Chad M Botz; Danelle H Murray; Melissa R Snyder; Jerry A Katzmann; Angela Dispenzieri; David L Murray Journal: J Proteome Res Date: 2014-02-11 Impact factor: 4.466
Authors: John R Mills; Mindy C Kohlhagen; Surendra Dasari; Patrick M Vanderboom; Robert A Kyle; Jerry A Katzmann; Maria A V Willrich; David R Barnidge; Angela Dispenzieri; David L Murray Journal: Clin Chem Date: 2016-08-18 Impact factor: 8.327